Protein-DNA selectivity is a central event in many biological processes. Type II restriction enzymes are ideal systems for studying selectivity, due to their high specificity and striking variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to proteins mediating site-specific recombination and DNA repair by excision. The long-term goals of this project are to understand the mechanisms by which type II restriction enzymes recognize and cleave DNA, and to design mutants with altered specificities. Our work is focused on the endonucleases Bam HI, Fok I, and Sfi I. We have determined structures of Bam HI at different stages of its catalytic pathway by X-ray crystallography. However, the critical structure that is missing for a complete understanding of specificity is that of a non-cognate DNA complex. Our specific goals are 1) to determine the structure of Bam HI bound to a non-cognate DNA site, 2) to determine the structure of the active site E113K mutant, and 3) to define the cleavage properties of the K205A mutant. Together, these studies will provide a complete, dynamic view of the sequence of events underlying sequence specific DNA cleavage. We have determined the structures of the complete Fok I endonuclease both with and without DNA. Fok I is an unusual bipartite enzyme that exists in solution as a monomer. A question that arises from our work is how the monomeric enzyme manages to cleave both strands? Our hypothesis is that Fok I dimerizes on DNA with the recognition domain of the second Fok I molecule accommodated on another DNA molecule (trans binding). Our specific goals are 1) to test this hypothesis by biophysical and biochemical methods, and 2) to use the bipartite nature of Fok I to create a novel hybrid enzyme capable of cleaving RNA at any pre-determined site. With Sfi I we now have cocrystals with DNA that diffract to 2.2 Angstrom units in resolution. Sfi I is a tetrameric enzyme that needs to interact with two DNA sites (either in cis or trans) in order to become activated for DNA cleavage. Our specific goals are 1) to determine the structure of the Sfi I/DNA complex using heavy atom derivatives, 2) to determine the structure of "free" Sfi I using molecular replacement methods, and 3) to determine the structures of cleavage intermediates. The Sfi I reaction provides a system for understanding long-range interactions on DNA.